Concise and Practical Asymmetric Synthesis of a Challenging Atropisomeric HIV Integrase Inhibitor.

A practical and efficient synthesis of a complex chiral atropisomeric HIV integrase inhibitor has been accomplished. The combination of a copper-catalyzed acylation along with the implementation of the BI-DIME ligands for a ligand-controlled Suzuki cross-coupling and an unprecedented bis(trifluoromethane)sulfonamide-catalyzed tert-butylation renders the synthesis of this complex molecule robust, safe, and economical. Furthermore, the overall synthesis was conducted in an asymmetric and diastereoselective fashion with respect to the imbedded atropisomer.

Preclinical profile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor.

BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3'-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 µM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

Discovery of a novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors.

A novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors were derived from a fragment-based approach using information from X-ray crystallographic analysis of NS5B-inhibitor complexes and iterative rounds of parallel synthesis. Structure-based drug design strategies led to the discovery of potent sub-micromolar inhibitors 11a-c and 12a-c from a weak-binding fragment-like structure 1 as a starting point.

From benzimidazole to indole-5-carboxamide Thumb Pocket I inhibitors of HCV NS5B polymerase. Part 1: indole C-2 SAR and discovery of diamide derivatives with nanomolar potency in cell-based subgenomic replicons.

Replacement of the benzimidazole core of allosteric Thumb Pocket 1 HCV NS5B finger loop inhibitors by more lipophilic indole derivatives provided up to 30-fold potency improvements in cell-based subgenomic replicon assays. Optimization of C-2 substitution on the indole core led to the identification of analogs with EC(50)<100 nM and modulated the pharmacokinetic properties of the inhibitors based on preliminary data from in vitro ADME profiles and in vivo rat PK.

Indole 5-carboxamide Thumb Pocket I inhibitors of HCV NS5B polymerase with nanomolar potency in cell-based subgenomic replicons (part 2): central amino acid linker and right-hand-side SAR studies.

In this part 2, new indole 5-carboxamide Thumb Pocket 1 inhibitors of HCV NS5B polymerase are described. Structure-activity relationships (SAR) were explored at the central amino acid linker position and the right-hand-side of the molecule in an attempt to identify molecules with a balanced overall profile of potency (EC(50)<100 nM), physicochemical properties and ADME characteristics.

Discovery of benzimidazole-diamide finger loop (Thumb Pocket I) allosteric inhibitors of HCV NS5B polymerase: Implementing parallel synthesis for rapid linker optimization.

Previously described SAR of benzimidazole-based non-nucleoside finger loop (Thumb Pocket I) inhibitors of HCV NS5B polymerase was expanded. Prospecting studies using parallel synthesis techniques allowed the rapid identification of novel cinnamic acid right-hand sides that provide renewed opportunities for further optimization of these inhibitors. Novel diamide derivatives such as 44 exhibited comparable potency (enzymatic and cell-based HCV replicon) as previously described tryptophan-based inhibitors but physicochemical properties (e.g., aqueous solubility and lipophilicity) have been improved, resulting in molecules with reduced off-target liabilities (CYP inhibition) and increased metabolic stability.

N-Acetamideindolecarboxylic acid allosteric 'finger-loop' inhibitors of the hepatitis C virus NS5B polymerase: discovery and initial optimization studies.

SAR studies at the N(1)-position of allosteric indole-based HCV NS5B inhibitors has led to the discovery of acetamide derivatives with good cellular potency in subgenomic replicons (EC(50) <200 nM). This class of inhibitors displayed improved physicochemical properties and favorable ADME-PK profiles over previously described analogs in this class.

An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus.

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

Discovery of BI 207524, an indole diamide NS5B thumb pocket 1 inhibitor with improved potency for the potential treatment of chronic hepatitis C virus infection.

The development of interferon-free regimens for the treatment of chronic HCV infection constitutes a preferred option that is expected in the future to provide patients with improved efficacy, better tolerability, and reduced risk for emergence of drug-resistant virus. We have pursued non-nucleoside NS5B polymerase allosteric inhibitors as combination partners with other direct acting antivirals (DAAs) having a complementary mechanism of action. Herein, we describe the discovery of a potent follow-up compound (BI 207524, 27) to the first thumb pocket 1 NS5B inhibitor to demonstrate antiviral activity in genotype 1 HCV infected patients, BILB 1941 (1). Cell-based replicon potency was significantly improved through electronic modulation of the pKa of the carboxylic acid function of the lead molecule. Subsequent ADME-PK optimization lead to 27, a predicted low clearance compound in man. The preclinical profile of inhibitor 27 is discussed, as well as the identification of a genotoxic metabolite that led to the discontinuation of the development of this compound.

Discovery of BI 224436, a Noncatalytic Site Integrase Inhibitor (NCINI) of HIV-1.

An assay recapitulating the 3' processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.

Discovery of the first thumb pocket 1 NS5B polymerase inhibitor (BILB 1941) with demonstrated antiviral activity in patients chronically infected with genotype 1 hepatitis C virus (HCV).

Combinations of direct acting antivirals (DAAs) that have the potential to suppress emergence of resistant virus and that can be used in interferon-sparing regimens represent a preferred option for the treatment of chronic HCV infection. We have discovered allosteric (thumb pocket 1) non-nucleoside inhibitors of HCV NS5B polymerase that inhibit replication in replicon systems. Herein, we report the late-stage optimization of indole-based inhibitors, which began with the identification of a metabolic liability common to many previously reported inhibitors in this series. By use of parallel synthesis techniques, a sparse matrix of inhibitors was generated that provided a collection of inhibitors satisfying potency criteria and displaying improved in vitro ADME profiles. "Cassette" screening for oral absorption in rat provided a short list of potential development candidates. Further evaluation led to the discovery of the first thumb pocket 1 NS5B inhibitor (BILB 1941) that demonstrated antiviral activity in patients chronically infected with genotype 1 HCV.

Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335).

C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A substantial contribution to the potency of these linear inhibitors arises from the introduction of a C8 substituent on the B-ring of the quinoline moiety found on the P2 of these inhibitors. The introduction of a C8 methyl group results not only in a modest increase in the cell-based potency of these inhibitors but more importantly in a much better pharmacokinetic profile in rats as well. Exploration of C8-substitutions led to the identification of the bromo derivative as the best group at this position, resulting in a significant increase in the cell-based potency of this class of inhibitors. Structure-activity studies on the C8-bromo derivatives ultimately led to the discovery of clinical candidate 29 (BI 201335), a very potent and selective inhibitor of genotype1 NS3 protease with a promising PK profile in rats.

Binding site characterization and resistance to a class of non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase.

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzimidazole 5-carboxamide and related compounds were found at three amino acid positions in the thumb domain: Pro(495) with substitutions to Ser, Leu, Ala, or Thr; Pro(496) substitutions to Ser or Ala; and a V499A substitution. Mutations at each of these positions conferred different levels of resistance to this drug class: the Pro(495) changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro(496) substitutions, and finally only minor shifts in potency with V499A. Combinations that include the benzimidazole 5-carboxamide polymerase inhibitors and compounds that bind other sites or other HCV targets, including HCV protease inhibitors, are complementary in cell culture models of HCV RNA replication at suppressing the emergence of resistant variants. This novel class of compounds and unique binding site expand the diversity of HCV antivirals currently under development and offer the potential to improve the treatment of chronic HCV infection.

Non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase: discovery and preliminary SAR of benzimidazole derivatives.

Benzimidazole 5-carboxamide derivatives from a combinatorial screening library were discovered as specific inhibitors of the NS5B polymerase of the hepatitis C virus (HCV). Initial hit-to-lead activities taking advantage of high-throughput parallel synthetic techniques, identified a 1,2-disubstituted benzimidazole 5-carboxylic acid scaffold as the minimum core for biological activity. Potent analogues in this series inhibit the polymerase at low micromolar concentrations and provide an attractive "drug-like" lead structure for further optimization and the development of potential HCV therapeutics.

Non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase: discovery of benzimidazole 5-carboxylic amide derivatives with low-nanomolar potency.

Optimization of benzimidazole 5-carboxamide derivatives previously identified as specific inhibitors of the NS5B polymerase of the hepatitis C virus (HCV) has led to the discovery of potent analogues that inhibit the enzyme at low-nanomolar concentrations. Greater than 800-fold improvement in potency from the original lead structure was achieved through the combined effects of conformational rigidification, molecular size extension and the identification of previously unexploited interactions. Furthermore, these inhibitors retain specificity for HCV polymerase relative to other viral and mammalian RNA polymerases.